Field of the Invention
This invention relates to a novel L-glutamic acid oxidase and its production.
More specifically, the present invention relates to an L-glutamic acid oxidase which exhibits a strong affinity and a high substrate specificity for L-glutamic acid, but has substantially no action on other amino acids and yet has a high stability, and to a microbiological method of production thereof.
Recently, an L-amino acid oxidase having a substrate specificity for L-glutamic acid has been found to be produced by cultivation of a microorganism belonging to the genus Streptomyces (hereinafter sometimes abbreviated as "S."), more specifically Streptomyces violascens (See Japanese Patent Laid-Open Publication No. 43685/1982). The physicochemical properties of the glutamic acid oxidase (hereinafter sometimes abbreviated as "known enzyme") as a protein have not yet been clarified, but the known enzyme is described to have enzymological properties as follows.
(1) Substrate specificity
When the velocity of enzymatic reaction for L-glutamic acid is given as 100, the known enzyme has a relative activity of 8.4 for L-glutamine and 6.8 for L-histidine, exhibiting substantially no activity for other amino acids.
(2) Optimum pH
pH 5-6
(3) pH stability
Stable in the range of pH 3.5-6.5 (37.degree. C., maintained for one hour)
(4) Temperature stability
Stable up to 50.degree. C. (maintained for 10 minutes)
(5) Influence of inhibitors
Substantially completely inhibited by mercury ions, copper ions and diethyldithiocarbamate.
The specification of the above Laid-Open Publication states that a liquid culture of the aforesaid microorganism is preferable for production of the known enzyme.
For utilization of the known enzyme for analysis of L-glutamic acid, various problems are involved. Specifically, although the known enzyme has a higher substrate specificity for L-glutamic acid as compared with other L-amino acid oxidases known in the art, it still exhibits clear activities for other amino acids as mentioned above, and therefore it cannot be used for specific quantitative determination of L-glutamic acid in the presence of these amino acids. Also, the known enzyme does not have a high pH stability and heat stability, and it cannot be considered to always have a good storage stability and stability during use as a reagent for analysis. Further, when copper ions exist in a sample to be analyzed, the activity of the known enzyme is markedly inhibited, whereby analysis may be considered to become difficult. Furthermore, the pH of reaction solutions employed in various clinical biochemical diagnostic analysis, especially in analysis of the activity of enzymes in blood, is usually around neutral, while the known enzyme will completely lose its activity at a pH of 7.5 when treated at 37.degree. C. for one hour. For this reason, it may be difficult to use the known enzyme in analysis around the neutral pH range.